DEVELOPMENT OF A CONTINUOUS FLUORESCENCE ASSAY FOR RHINOVIRUS-14 3C PROTEASE USING SYNTHETIC PEPTIDES

Rhinovirus 3C protease is an attractive target for therapeutic interve ntion owing to its important role in virion maturation and infectivity . In order to facilitate the identification of potential 3C protease i nhibitors, we have developed a continuous fluorescence assay using 5-[ (2-aminoethyl)amino]naphthalene-1-sulphonic acid (Edans) as a fluoresc ent donor and 4-(4-dimenthylaminophenyl-azo)benzoic acid (Dabcyl) as a quenching acceptor. Several fluorogenic peptide substrates for 3C pro tease were synthesized by both solution chemistry and solid phase pept ide synthesis. One of the synthetic Edans/Dabcyl substrates, with an a mino acid sequence derived from the 2C/3A site of the virus polyprotei n, yielded a 24-fold increase in fluorescence intensity after 3C cleav age. Data regarding substrate cleavage kinetics, assay sensitivity and optimization are presented. The application of this assay to the eval uation of 3C protease inhibitors is also shown.